This invention relates to an herbal formulation useful for blocking of 5 lipoxygenase activity leading to the inhibition of leukotrine synthesis, suppression of IL4 production and enhancement of IFN gamma release. More particularly this invention describes the process for the preparation of extracts from the leaves or any other plant parts of plants M. koenigii and P. betel and their selective blending to evaluate biological response in order to establish their role for the treatment and remedy of bronchial respiratory troubles.
Respiratory problems consist of mild to extremely severe trouble of breathing along with the other discomforts such as wheezing, coughing, chest tightness and the like. In spite of precautionary measure, public awareness campaign and monitoring system, population having respiratory trouble is on the rise all over the world. This is more true in western advanced countries, specially among the children. With M. koenigii leaf preparation, the relief and possible cure of asthma has been demonstrated in a large number of cases (Unpublished PCT Patent application PCT/IN/00102). Two-prong strategy was adapted in the present invention. The respiratory disease is the result of pathophysiological symptoms arising out of aberration of immune system. The immediate symptom includes bronchial constriction, inflammation of respiratory tract and closing of air way by mucus secretion. The symptomatic drugs provide relief by temporary relaxation of the distressed symptoms.
This is to state that the Murrya koenigii extract combined with extract obtained from piper betel has shown surprising results. According to the earlier filed unpublished PCT patent application, the betel leaf extract showed increase in IFN-xcex3 from Th1 type cells and decrease of IL-4 from Th2 type cells. This clearly is an effective measure in treating asthma as shifting of Th2 type response towards Th1 type will obviously reduce the release of IgE from mast cells which is a major manipulator of asthmatic condition. Hence the applicants earlier finding followed by the filing of PCT patent application PCT/INOO/00127 is in direct consonance with the present filing on combined extracts from any plant parts of M. koenigii and P. betel as a new medicine for treating atopic Asthma.
The root cause for respiratory problems is not well addressed by the developers of symptomatic drugs. With the advent of current knowledge, it is now well accepted that leukotrienes are found to be the main player in developing symptoms of respiratory problems.
The major symptoms of respiratory problem can be divided into early and late responses. The early response occurs within minutes of allergen exposure and involves primarily mediators such as histamine, leukotrienes and prostaglandin D2. The effects of these mediators result in bronchoconstriction and accumulation of mucus. The late response occurs hours later and involves additional mediators including IL-4, IL-5, IL-6, and TNF-alpha, eosinophils chemotactic factor (ECF) and platelet activating factor (PAF). The overall effect of these mediators is to recruit inflammatory cells including eosinophils and neutrophils. These cells are capable of causing significant tissue injury by releasing toxic enzymes. These events lead to occlusion of bronchial lumen with mucus protein, and cellular debris, thus thickening of basement membrane, fluid build up and hypertrophy of the bronchial smooth muscle. A mucus plug often forms and adheres to the bronchial wall. The mucus plug contains clusters of detached epithelial cells fragments, eosinophils, some neutrophils and spirals of bronchial tissue known as Curschmann""s spiral (Immunology, J. Kuby; W.H. Freeman and Co., New York; 3rd edition 1997). In view of the current mechanisms regarding manifestation of bronchial asthma/ bronchial respiratory problems, modem strategy for drug development stressed the following approaches:
These include i) inhibition of leukotriene synthesis via blocking the 5-lipoxygenase-enzyme activity (Leqqis RA et al New England J. Med. 323:,645,1990). The formation of leukotrienes originates from the oxidation of arachidonic acid, hence inhibition of this reaction leads to the inhibition of leukotriene synthesis. Besides, leukotrine receptors antagonists have also been introduced as anti leukotriene therapy for asthma/respiratory problems (Tien F. C., Medical J. Aust. 171: 378,1999). Currently licensed drug zelutin, based on inhibition of arachidonic acid oxidation has already been introduced exclusively in the US market. However, its use is limited by hepatotoxicity. Children below 14 years are not recommended for this drug. Moreover patients taking other drugs need to be surveyed when taking zelutin as anti-asthma drug. More over patients taking other drugs need to be surveyed when taking zelutin, as anti-asthma drug. ii) the neutralization of IgE either by anti-IgE antibody (humanized) or by blocking the high affinity IgE receptor, Fcxcex5R-I (Heusser C, Jardiu P. Current Oppinio Immunol., 9: 805.1999). Since suppression of Th2 cytokines leads to decrease in IgE production, additional approach is based on the inhibition of these Th2 cytokine synthesis and enhancement of Th1 cytokine formation (Chung K. F.; Barens P. J. Thorax 54: 825, 1999).
In contrast, M. koenigii based anti asthmatic preparation has been tried on children as young as 7 years old and octogenarian as old as 80 years and above without any adverse reaction.
The extract of M. koenigii leaf was therefore examined for their potential to inhibit the production of leukotrienes. Additionally, the extract was also examined for shifting of Th2 response towards to Th1 type. Th1 and Th2 response were measured by 7-interferon (Th 1) and IL-4 (Th2) production respectively. M. koenigii extract showed inhibition of 5-lipoxygenase mediated arachidonic acid oxidation signifying blockage of leukotriene synthesis and marginal effect on xcex3-interferon production. Importantly, M. koenigii extract drastically reduces IL-4 synthesis. On the other hand, P. betel leaf extracts resulted in facilitating of the shift from Th2 type response to Th1 type. In other words, a man having average skill in the art cannot just combine these two to produce inference of the combination of the ingredients. This combination is a novel combination and the combined effects of the combination are surprising and unexpected. Thus, a blend of extracts from these two plants, M. koenigii and P. betel predominantly inhibited leukotriene synthesis and shifted Th2 response towards Th1 type and therefore is proposed as a unique synergistic formulation for treatment, relief and remedy of bronchial respiratory problems. Thus, the two pronged strategy is the major objective of this new formulation and can be considered as the best modality of treatment for patients suffering from bronchial respiratory problems.
The main objective of the invention is to provide a new pharmaceutical formulation comprising combination of extracts derived from plant parts leaves of Murraya koenigii and Piper betel or any of the plant parts thereof.
Another object of the invention is to provide a new pharmaceutical formulation comprising combination of extracts derived from Murraya koenigii and Piper betel leaves.
Yet another objective of the present invention is to provide a process for the preparation of extract from the leaves or any other plant parts of M. koenigii and P. betel useful for relief, treatments and remedy of respiratory problem.
Yet another objective of the present invention is to provide a simplified method of extract preparation from leaves or any other plant parts of M. koenigii and P. betel possessing biological activities relevant to treatment, relief and remedy of respiratory problems.
Yet another objective of the present invention is to provide a simplified fast and inexpensive process for the preparation of combination of extracts possessing biological activities relevant to treatments, relief and remedy of respiratory problems.
Yet another objective of the present invention is to provide a herbal preparation comprising of extracts derived from M. koenigii and P. betel leaves or extracts obtained from other plant parts of M. Koenigii and P. betel, wherein the said extracts being highly compatible for human consumption and capable for being used for the treatments, relief and remedy of respiratory problem.
Yet another objective of the present invention is to examine each of the extracts obtained from M.koenigii and P. betel leaves or their other plants parts individually and in combination on the inhibitory activity of 5-lipoxygenase-mediated Arachidonic acid oxidation and inducing the shift of Th2 type response towards Th1 type.
Yet another objective of the present invention is to assay 5-lipoxygenase mediated Arachidonic acid oxidation in an ex vivo human whole blood system in the presence of M. koenigii and P. betel leaves extracts or their other plant part extracts individually and in combination.
Yet another objective of the present invention is to assay the 5-lipoxygenase mediated Arachidonic acid oxidation in an ex vivo system with enriched human polymorphonuclear neutrophils (PMN) in the presence of M. koenigii and P. betel leaves extracts or their other plant part extracts, individually and in combination.
Yet another objective of the present invention is to analyze the production of intercellular cytokines by flow cytometry.
Yet another objective of the present invention is to provide intracellular IFN-gama and IL-4 profile as known markers for Th1 and Th2 type response respectively in ex vivo human whole blood with the extracts from M. koenigii and P. betel leaves individually and in combination
In accordance the present invention provides an herbal formulation useful for blocking of 5 lipoxygenase activity leading to the inhibition of leukotrine synthesis, suppression of IL4 production and enhancement of IFN gamma release
Also, the present invention provides an herbal formulation for the treatment and remedy of bronchial respiratory difficulties. More particularly this invention provides a process for the preparation of extracts from the leaves or any other plant parts or seeds of plants M. koenigii and P. betel and their selective blending to evaluate biological response in order to establish their role for the treatment and remedy of bronchial respiratory troubles.
The present invention also provides a herbal formulation useful for blocking of 5 lipoxygenase activity leading to the inhibition of leukotrine synthesis, suppression of IL4 production and enhancement of IFN gamma release. Also, the present invention provides a method of treating a subject for bronchial respiratory conditions, said method comprising administering to the subject effective amount of extracts obtained from the leafs or any other plant parts of Piper betel and Murrya koenigii or lyophilized extracts of Piper betel (PB) and Murrya koenigii (M.K) or a formulation comprising lyophilized extracts.
Accordingly, the present invention provides a pharmaceutical formulation useful as leukotrine and IL-4 synthesis inhibitor and Th1 type immunomodulator, said formulation comprising effective amount of extract or lyophilised extract obtained from the leaves or any other plant parts of Piper betel (PB) and Muriva koenigii (M.K) associated with or in combination with pharmaceutically acceptable additives.
In an embodiment of the present invention, the extract obtained from Piper betel is used as Th1 type immunomodulator and the extract obtained from Murrya koenigii (M.K) is used for leukotrine synthesis inhibitor.
In another embodiment of the invention provides a the formulation comprising effective amount of lyophilized extracts of P.betel and M.koenigii optionally associated with or in combination with a pharmaceutically acceptable carrier wherein, the carrier is selected in such a manner it does not interfere with the activity of lyophilized extracts of piper betel and M. koenigii extracts
In another embodiment of the invention relates to the selection of additive. The additive is selected from nutrients such as proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, starch-gelatin paste and/or pharmaceutically acceptable carriers, excipient, diluent or solvent.
Still another embodiment, the aqueous extract, lyophilized product or the formulation is administered inhalation, oral, intravenous, intramuscular, subcutaneous route or any other suitable routes and the oral route is in the form of capsule, syrup, concentrate, powder or granules.
Still another embodiment relates to the amount of aqueous extract, lyophilized product or the formulation is administered by intravenous route is less than the oral route.
In yet another embodiment, the ratio of betel extract to the M.K extract is in the range between 1:1.0 to 1:5.0.
In yet another embodiment, the proportion of M.K extract or lyophilized extract is equal to or greater than the amount of betel extract or lyophilised to the M.K extract.
In yet another embodiment, the formulation comprising the betel extract and M.K extract is administered at a dosage level between 1 to 20 mg/kg of body weight up to three times a day for a period at least 4 weeks.
In yet another embodiment, the formulation comprising the betel extract and M.K extract is administered for a period of at least 4 weeks and up to three months depending upon the conditions.
In yet another embodiment relates to a method for treating animals and human beings.
One more embodiment relates to a pharmaceutical formulation useful for the treatment of bronchial respiratory conditions, said formulation comprising effective amount of lyophilized extracts obtained from the leafs or any other plant parts of Piper betel (PB) and Murrya koenigii M.K) optionally associated with or in combination with pharmaceutically acceptable additive.
Another embodiment of the present invention, the plant parts used for preparing the extracts or lyophilized extracts is selected from leafs, stems, barks, fruits, seeds or any other parts of the plants Piper betel (PB) and Murrya koenigii (M.K)
In one more embodiment relates to the use of formulation for shifting Th2 type response to Th1 type response.
Yet another embodiment relates to the use of formulation for inhibiting 5-lipooxygenase mediated Arachidonic acid oxidation in neutrophils enriched fraction of whole blood.
Yet another embodiment relates to the use of formulation for enhancing IFN-gamma and reducing IL-4 response in ex-vivo human whole blood.
Yet another embodiment relates to the use of formulation for enhancing IFN-gamma response in ex vivo human whole blood mononuclear (PMN)
Yet another embodiment relates to the use of formulation for reducing IL-4 response in human peripheral whole blood mononuclear cells.
In another embodiment relates to use of piper betel leaf extract and M.K leaf extract or extract obtained from any other plant parts of plants Piper betel and M.K for the treatment of bronchial respiratory conditions.
Yet another embodiment relates to use of betel leaf extract and M.K leaf extract or extract obtained from any other plant parts of plants Piper betel and M.K for shifting Th2 response to Th1 response.
Yet another embodiment relates to use of betel leaf extract and M.K leaf extract or extract obtained from any other plant parts of plants Piper betel and M.K for inhibiting 5-lipooxygenase mediated Arachidonic acid oxidation in neutrophils enriched fraction of whole blood.
Yet another embodiment relates to use of betel leaf extract and M.K leaf extract or extract obtained from any other plant parts of plants Piper betel and M.K for enhance IFN-gamma and reduce IL-4 response in ex vivo human whole blood.
Yet another embodiment relates to use of betel leaf extract and M.K leaf extract or extract obtained from any other plant parts of plants Piper betel and M.K for enhance IFN-gamma response in ex vivo human whole blood mononuclear (PMN)
Yet another embodiment relates to use of betel leaf extract and M.K leaf extract or extract obtained from any other plant parts of plants Piper betel and M.K for reducing IL-4 response in human peripheral whole blood mononuclear cells.
In an embodiment of the present invention, the extracts obtained from the plants M. koenigii and P. betel are mixed with freshly drawn human blood.
In an another embodiment of the present invention, the cells in an ex vivo human blood system activated with calcium inophor or the likes.
In an another embodiment of the present invention, extracts from M. koenigii and P. betel leaf are blended by the technique known in the art.
In an another embodiment of the present invention, the leaf extracts are made from fresh or sun/shade dried of M. koenigii and P. betel. 
In an another embodiment of the present invention, plant material are used for extraction with appropriate solvent such as methanol or water or buffer in a percolator or the equipment known in the art.
In an another embodiment of the present invention, the extracts of M. koenigii and P. betel are concentrated under reduced pressure to save active principle.
In yet another embodiment of the present invention, the concentrates are lyophilised to remove reduced water and other residual solvent.
In yet another embodiment of the present invention, the fraction found active as inhibitor of 5-lipoxygenase mediated Arachidonic acid oxidation in an ex vivo whole human blood are selected for formulation.
In yet another embodiment of the present invention, the extracts of M. koenigii and P. betel found active for shifting from Th2 type response to the Th1 response are selected for formulation.
In yet another embodiment of the present invention, the extracts of M. koenigii and P. betel inhibited 5-lipoxygenase mediated Arachidonic acid oxidation in neutrophils enriched fraction of whole blood.
In yet another embodiment of the present invention, the blended extract of M. koenigii and P. betel is found to enhance IFN-gama and reduce IL-4 response in ex vivo human whole blood.
In yet another embodiment of the present invention, the extract obtained from P. betel is found to enhancc IFN-gama response in ex vivo human blood mononuclear (PMN).
In yet another embodiment of the present invention, the formulated extract prepared from the leaf extracts of M. konigii and P. betel is found to reduce IL-4 response in human peripheral blood mononuclear cells.
In yet another embodiment of the present invention, the leaf extracts of M. konigii and P. betel were found as inhibitors of 5-lipoxygenase mediated arachidonic acid oxidation assayed after incubation of whole blood and lysing the cells at the time of assay were chosen for formulation.